In basic research, one goal is to understand how genes are distributed within populations and how expression of those genes leads to phenotypic differences. This information has the potential to become a powerful tool for predicting human health trends and has been a driving force behind the search for genetic markers for human disease.
Over the years, many biochemical techniques have been introduced for analyzing the presence and/or amount of a biomolecule in a sample. As examples, a number of organic stains have been adapted for the detection of electrophoretically separated proteins, including Bromphenol Blue, Coomassie Blue, Fast Green (Food Green 3) and Amido Black (Acid Black 1). (See Durrem, J. Am. Chem. Soc. 72:2943 (1950), Grassman and Hannig, Z. Physiol. Chem. 290:1 (1952), Fazekas De St. Groth et al., Biochim. Biophys. Acta 71:377 (1963), and Meyer and Lamberts, Biochim. Biophys. Acta 107:144 (1965)). Fluorescent stains, such as fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-Furanone (MDPH), are also used to detect proteins (See Ragland et al., Anal. Biochem. 59:24 (1974) and Pace et al., Biochem. Biophys. Res. Commun. 57:482 (1974)). A sensitive technique for staining proteins is silver staining. (See Merril et al., Proc. Natl. Acad. Sci. USA 76:4335 (1979) and Switzer et al., Anal. Biochem. 98:231 (1979)). While these techniques may be useful to resolve total protein in a sample, they are limited in their usefulness to detect a specific protein in a heterogeneous population of proteins.
The detection of specific proteins in a sample can be accomplished by techniques including Western blot, immunoprecipitation, enzyme-linked immunoassay (ELISA), and sandwich assays. These techniques typically use radioactivity, fluorescence, and chemiluminescence to label or mark an antibody or other protein which binds to the target protein and thereby identifies the presence and/or location of the target. Depending on the quality of the antibody and the label used, the sensitivity of detection and non-specific binding varies.
Radioactivity, fluorescence, and chemiluminescence are also commonly used for the detection of specific nucleic acid sequences in a sample. Hybridization techniques, such as Southern and Northern blotting, are frequently employed to detect the presence of polymorphisms in a nucleic acid sample. In nucleic acid hybridization, for example, a radioactive label (e.g. 32P or 35S) is incorporated into an oligonucleotide probe which complements a target nucleic acid, and hybridization with the target is accomplished at a specific salt concentration and temperature. (See e.g. Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (1989)).
Southern et al. has used nucleic acid hybridization by setting up an array of oligonucleotides on plastic and glass, probing with a radioactive oligonucleotide, and detecting the presence of a target nucleic acid with a PhosphorImager. (See Southern, E. M. et al., Nucleic Acids Res 22, 1368-1373 (1994)). The PhosphorImager instrument, an expensive laser based optical system, and clean image-ready phosphor screens are needed for each sample read, making the system both cumbersome and very expensive. In addition, radioactive probes have a short shelf life (T2=days to months) and require tight inventory control in a licensed facility. Although some companies are currently performing genetic screening using this method, the cost is prohibitive for most diagnostic procedures.
Others in the field are pursuing methods more predisposed to automation in hopes of enabling the rapid screening of a sample for a number of sequences. As one example, Affymetrix (Santa Clara, Calif.) has described a system which performs on-chip hybridization. (See Kreiner, T., American Laboratory March:39-43 (1996). In this system, oligonucleotides are arrayed in 90×90:m cells with 107 oligonucleotides per cell, with 20,000 probe cells on each chip. This is annealed with fluorescence-labeled probes, and detection is carried out using a 488 nm Argon laser (8:m shot size) as a excitation source and a photomultiplier tube to detect the fluorescence emission. To read the chip, an optical system consisting of a dichromic mirror, scanning head, routing mirror and a confocal optical system are employed. One significant problem with this approach is non-specific background. Several natural occurring molecules either contribute to or quench the fluorescent signal, making this technique prone to a background noise which prevents this system from achieving highly sensitive nucleic acid detection.
Chemiluminescence is another marker employed to detect biomolecules. Chemiluminescence uses an enzyme coupled to the probe which catabolizes a chemical substrate to generate a photon. (See Bronstein, et al., BioTechniques 8:310-313 (1990)). Chemiluminescence nucleic acid hybridization assays may use a high performance, low-light-sensitive charge coupled device (CCD) camera to image the light emission from the chemical reaction. Often the camera is controlled by a personal computer and the images are archived on diskettes. While the CCD cameras are robust, CCD based systems do not have the sensitivity of film and the reagents have a one-year shelf life when stored at 4° C. (Tropix Inc.). As with fluorescence detection approaches, this approach is limited by background noise caused by naturally occurring enzymes or compounds contributing to the signal.
As the secrets of genomic regulation and the biosynthesis of enzymes, receptors, and ligands involved in human disease unfold, the need for detection techniques which provide a high degree of specificity and sensitivity with minimal background noise, while minimizing cost and handling issues, is manifest. In view of the foregoing, and notwithstanding the various efforts exemplified in the prior art, there remains a need for novel compositions, methods, and systems for highly sensitive biomolecule detection.